A dual-plasmid CRISPR/Cas9-based method for rapid and efficient genetic disruption in Mycobacterium abscessus.

Mycobacterium abscessus is increasingly recognized for causing infections that are notoriously difficult to treat, owing to its large arsenal of intrinsic antibiotic resistance mechanisms. Tools for the genetic manipulation of the pathogen are critical for enabling a better understanding of M. abscessus biology, pathogenesis, and antibiotic resistance mechanisms. However, existing methods are largely recombination-based, which are relatively inefficient. Meanwhile, CRISPR/Cas9 has revolutionized the field of genome editing including its recent adaptation for use in mycobacteria. In this study, we report a streamlined and efficient method for rapid genetic disruptions in M. abscessus. Harnessing the CRISPR1 loci from Streptococcus thermophilus, we have developed a dual-plasmid workflow that introduces Cas9 and sgRNA cassettes in separate steps but requires no other additional factors to engineer mutations in single genes or multiple genes simultaneously or sequentially using multiple targeting sgRNAs. Importantly, the efficiency of mutant generation is several orders of magnitude higher than reported for homologous recombination-based methods. This work, thus, reports the first application of CRISPR/Cas9 for gene editing in M. abscessus and is an important tool in the arsenal for the genetic manipulation of this human pathogen. IMPORTANCE: Mycobacterium abscessus is an opportunistic pathogen of increasing clinical importance due to its poor clinical outcomes and limited treatment options. Drug discovery and development in this highly antibiotic-resistant species will require further understanding of M. abscessus biology, pathogenesis, and antibiotic resistance mechanisms. However, existing methods for facile genetic engineering are relatively inefficient. This study reports on the first application of CRISPR/Cas9 for gene editing in M. abscessus using a dual-plasmid workflow. We establish that our method is easily programmable, efficient, and versatile for genetic disruptions in M. abscessus. This is a critical advancement to facilitating targeted gene function studies in this emerging pathogen.

Zafirlukast induces DNA condensation and has bactericidal effect on replicating Mycobacterium abscessus.

Mycobacterium abscessus infections are emerging in cystic fibrosis patients, and treatment success rate in these patients is only 33% due to extreme antibiotic resistance. Thus, new treatment options are essential. An interesting target could be Lsr2, a nucleoid-associated protein involved in mycobacterial virulence. Zafirlukast is a Food and Drug Administration (FDA)-approved drug against asthma that was shown to bind Lsr2. In this study, zafirlukast treatment is shown to reduce M. abscessus growth, with a minimal inhibitory concentration of 16 µM and a bactericidal concentration of 64 µM in replicating bacteria only. As an initial response, DNA condensation, a known stress response of mycobacteria, occurs after 1 h of treatment with zafirlukast. During continued zafirlukast treatment, the morphology of the bacteria alters and the structural integrity of the bacteria is lost. After 4 days of treatment, reduced viability is measured in different culture media, and growth of M. abscessus is reduced in a dose-dependent manner. Using transmission electron microscopy, we demonstrated that the hydrophobic multilayered cell wall and periplasm are disorganized and ribosomes are reduced in size and relocalized. In summary, our data demonstrate that zafirlukast alters the morphology of M. abscessus and is bactericidal at 64 µM. The bactericidal concentration of zafirlukast is relatively high, and it is only effective on replicating bacteria but as zafirlukast is an FDA-approved drug, and currently used as an anti-asthma treatment, it could be an interesting drug to further study in in vivo experiments to determine whether it could be used as an antibiotic for M. abscessus infections.

Efflux pump effects on levofloxacin resistance in Mycobacterium abscessus.

Mycobacterium abscessus (M. abscessus) inherently displays resistance to most antibiotics, with the underlying drug resistance mechanisms remaining largely unexplored. Efflux pump is believed to play an important role in mediating drug resistance. The current study examined the potential of efflux pump inhibitors to reverse levofloxacin (LFX) resistance in M. abscessus. The reference strain of M. abscessus (ATCC19977) and 60 clinical isolates, including 41 M. abscessus subsp. abscessus and 19 M. abscessus subsp. massilense, were investigated. The drug sensitivity of M. abscessus against LFX alone or in conjunction with efflux pump inhibitors, including verapamil (VP), reserpine (RSP), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), or dicyclohexylcarbodiimide (DCC), were determined by AlarmarBlue microplate assay. Drug-resistant regions of the gyrA and gyrB genes from the drug-resistant strains were sequenced. The transcription level of the efflux pump genes was monitored using qRT-PCR. All the tested strains were resistant to LFX. The drug-resistant regions from the gyrA and gyrB genes showed no mutation associated with LFX resistance. CCCP, DCC, VP, and RSP increased the susceptibility of 93.3% (56/60), 91.7% (55/60), 85% (51/60), and 83.3% (50/60) isolates to LFX by 2 to 32-fold, respectively. Elevated transcription of seven efflux pump genes was observed in isolates with a high reduction in LFX MIC values in the presence of efflux pump inhibitors. Efflux pump inhibitors can improve the antibacterial activity of LFX against M. abscessus in vitro. The overexpression of efflux-related genes in LFX-resistant isolates suggests that efflux pumps are associated with the development of LFX resistance in M. abscessus.

Mycobacterium Infection of Abscess with Hemoptysis as the Initial Manifestation.

BACKGROUND: Mycobacterium abscessus is a new pathogen in recent years, which belongs to non-tuberculosis mycobacterium. Mycobacterium abscessus is widely involved in many nosocomial infections and secondary aggravation of genetic respiratory diseases. Mycobacterium abscessus is naturally resistant to most antibiotics and is difficult to treat. We report a case of mycobacterium abscessus infection with hemoptysis as the first manifestation. METHODS: Bronchoscopy, next-generation sequencing (NGS). RESULTS: Acid-fast staining of bronchoscopic lavage fluid showed that a small amount of acid-fast bacilli could be seen. NGS test showed the presence of Mycobacterium abscess, sequence number 137 (reference range ≥ 0), and symptomatic treatment against non-tuberculosis mycobacteria. CONCLUSIONS: For the follow-up infection of patients with hemoptysis, the treatment effect of antibiotics is not good, so the pathological tissue should be obtained by bronchoscopy or percutaneous lung biopsy in time, and the diagnosis should be confirmed by NGS if necessary.

Three Cases of Non-Tuberculosis Mycobacterium Skin Infection Outbreak in Beauty Institutions.

BACKGROUND: From June 2021 to July 2021, our hospital confirmed 3 cases of Mycobacterium infection in skin abscesses. All 3 patients underwent thread embedding and weight loss surgery at the same informal beauty institution, with a history of silk protein injection. None of the patients had any other underlying diseases or surgical history. Symptoms and signs show that the disease is acute and the course of the disease is short. All patients have found subcutaneous masses in different parts of the body. In most cases, the masses show redness and swelling, and some of the masses are accompanied by tenderness, wave sensation, and rupture. After some of the masses rupture, purulent secretions can be seen. METHODS: The pus secreted by the skin lesions of the three patients were cultured to a single bacterium, which was identified by MALDI-TOF MS. Multiple locus sequence typing (MLST) was performed using three specific genes (hsp65, rpoB, and secA1) and seven housekeeping genes (argH, cya, glpK, gnd, murC, pta, and purH). The results were queried through the MLST database of Mycobacterium abscess. RESULTS: All three strains of bacteria were Mycobacterium abscess type ST279 massiliense subtype. Three antibacterial drugs including cefmetazole, amikacin, and clarithromycin were administered in combination with 5-aminolevulinic acid photodynamic therapy (ALA-PDT). After 3 - 6 months, there was no obvious redness or swelling in the surrounding tissues of the wound, and no obvious purulent secretions were observed. All patients were cured and discharged from the hospital. After a follow-up of six months, there was no recurrence of the lesions. CONCLUSIONS: Medical institutions must strictly follow infection control guidelines and take preventive measures to prevent such incidents from happening again. ALA-PDT as a combination therapy for nontuberculous Mycobacterium (NTM) skin infections can improve treatment efficacy and shorten antibiotic usage time.

Antibiotic resistance and genomic characterization of Mycobacterium abscessus complex isolates from patients with pulmonary tuberculosis in Iran: A multi-center study (2010-2021).

One hundred and eighteen sputum specimens suspected of Mycobacterium abscessus infection were collected. Species level identification of M. abscessus was performed by rpoB sequencing. Clonality analysis was done by multilocus sequence typing (MLST) for M. abscessus. Antibiotic susceptibility testing was performed for clarithromycin, amikacin, ciprofloxacin and moxifloxacin. Altogether 128 isolates were obtained and were subjected to rpoB gene sequencing for definite identification. Among them 59 were identified as M. abscessus, and these included 22 (37.28%) isolates of M. abscessus subsp. abscessus, 22 (37.28%) isolates of M. abscessus subsp. massiliense, and 15 (25.42%) isolates of M. abscessus subsp. bolletii. All 59 M. abscessus complex isolates were analyzed by MLST in this study. Certain sequence types (STs) were identified among the 59 isolates and were specific for each subspecies. Two STs (ST40 and ST33) were specific to M. abscessus subsp. abscessus, one ST (ST20) was specific to M. abscessus subsp. bolletii, and one ST (ST15) was specific to M. abscessus subsp. massiliense. In antibiotic resistance, clarithromycin susceptibility testing of 22 M. abscessus subsp. abscessus strains detected 15 (68.18%) resistant strains, while among 22 M. abscessus subsp. massiliense strains 5 (22.72%) exhibited resistance, and among 15 M. abscessus subsp. bolletii 8 (53.33%) were resistant. Our study revealed a significant level of antibiotic resistance in isolates of the M. abscessus complex.

A simplified and efficient method for single-step, targeted gene deletion in mycobacteria.

Targeted gene deletion in mycobacteria remain complicated, requiring expertise and multiple steps. Here we present a single-step, easy to understand and perform method for targeted gene deletion. Using this method, we successfully deleted several genes in both M. smegmatis and M. abscessus. We believe this method will facilitate molecular research of mycobacteria and make it accessible to a greater number of researchers throughout the world.

Gene knock-out in Mycobacterium abscessus using Streptococcus thermophilus CRISPR/Cas.

The CRISPRi system using dCas9Sth1 from Streptococcus thermophilus developed for Mycobacterium tuberculosis and M. smegmatis was modified to allow gene knock-out in M. abscessus. Efficacy of the knock-out system was evaluated by applying deletions and insertions to the mps1 gene. A comparative genomic analysis of mutants and wild type validated the target specificity.

Genetic stability of Mycobacterium abscessus during antibiotic treatment.

OBJECTIVES: Genetic changes in Mycobacterium abscessus during antibiotic treatment are not fully understood. This study aimed to investigate the genetic changes in M. abscessus in patients receiving antibiotic treatment, and their clinical implications. METHODS: Pretreatment and 12-month post-treatment M. abscessus isolates were obtained from patients with M. abscessus pulmonary disease. Isolates from each time point were separated into six groups based on their distinctive morphological characteristics. Twenty-four isolates, comprising 12 from patient A exhibiting progressive disease and 12 from patient B demonstrating stable disease, underwent sequencing. Subsequently, minimal inhibitory concentrations (MICs) for the administered antibiotics were measured. RESULTS: Persistent infection with a single strain was observed in patients A and B. During 12 months of treatment, MICs for administered drugs did not generally change over time in either patient and single nucleotide variations (SNV) associated with antimicrobial resistance (rrl, rrs, erm(41), gyrA, gyrB, whiB7 and hflX) were not mutated. Although not significant, 47 and 52 non-synonymous SNVs occurred in M. abscessus from patients A and B, respectively, and the accumulation of these SNVs differed in patients A and B, except for five SNVs. The most variable positions were within a probable NADH-dependent glutamate synthase gene and a putative YrbE family protein gene in patients A and B, respectively. CONCLUSIONS: Persistent infections by a single strain of M. abscessus were observed in two patients with different clinical courses. Genetic changes in M. abscessus during antibiotic treatment were relatively stable in these patients. CLINICAL TRIALS IDENTIFIER: NCT01616745 (ClinicalTrials.gov ID).

Lsr2, a pleiotropic regulator at the core of the infectious strategy of Mycobacterium abscessus.

Mycobacterium abscessus is a non-tuberculous mycobacterium, causing lung infections in cystic fibrosis patients. During pulmonary infection, M. abscessus switches from smooth (Mabs-S) to rough (Mabs-R) morphotypes, the latter being hyper-virulent. Previously, we isolated the lsr2 gene as differentially expressed during S-to-R transition. lsr2 encodes a pleiotropic transcription factor that falls under the superfamily of nucleoid-associated proteins. Here, we used two functional genomic methods, RNA-seq and chromatin immunoprecipitation-sequencing (ChIP-seq), to elucidate the molecular role of Lsr2 in the pathobiology of M. abscessus. Transcriptomic analysis shows that Lsr2 differentially regulates gene expression across both morphotypes, most of which are involved in several key cellular processes of M. abscessus, including host adaptation and antibiotic resistance. These results were confirmed through quantitative real-time PCR, as well as by minimum inhibitory concentration tests and infection tests on macrophages in the presence of antibiotics. ChIP-seq analysis revealed that Lsr2 extensively binds the M. abscessus genome at AT-rich sequences and appears to form long domains that participate in the repression of its target genes. Unexpectedly, the genomic distribution of Lsr2 revealed no distinctions between Mabs-S and Mabs-R, implying more intricate mechanisms at play for achieving target selectivity.IMPORTANCELsr2 is a crucial transcription factor and chromosome organizer involved in intracellular growth and virulence in the smooth and rough morphotypes of Mycobacterium abscessus. Using RNA-seq and chromatin immunoprecipitation-sequencing (ChIP-seq), we investigated the molecular role of Lsr2 in gene expression regulation along with its distribution on M. abscessus genome. Our study demonstrates the pleiotropic regulatory role of Lsr2, regulating the expression of many genes coordinating essential cellular and molecular processes in both morphotypes. In addition, we have elucidated the role of Lsr2 in antibiotic resistance both in vitro and in vivo, where lsr2 mutant strains display heightened sensitivity to antibiotics. Through ChIP-seq, we reported the widespread distribution of Lsr2 on M. abscessus genome, revealing a direct repressive effect due to its extensive binding on promoters or coding sequences of its targets. This study unveils the significant regulatory role of Lsr2, intricately intertwined with its function in shaping the organization of the M. abscessus genome.

Orphan response regulator NnaR is critical for nitrate and nitrite assimilation in Mycobacterium abscessus.

Mycobacterium abscessus (Mab) is an opportunistic pathogen afflicting individuals with underlying lung disease such as Cystic Fibrosis (CF) or immunodeficiencies. Current treatment strategies for Mab infections are limited by its inherent antibiotic resistance and limited drug access to Mab in its in vivo niches resulting in poor cure rates of 30-50%. Mab's ability to survive within macrophages, granulomas and the mucus laden airways of the CF lung requires adaptation via transcriptional remodeling to counteract stresses like hypoxia, increased levels of nitrate, nitrite, and reactive nitrogen intermediates. Mycobacterium tuberculosis (Mtb) is known to coordinate hypoxic adaptation via induction of respiratory nitrate assimilation through the nitrate reductase narGHJI. Mab, on the other hand, does not encode a respiratory nitrate reductase. In addition, our recent study of the transcriptional responses of Mab to hypoxia revealed marked down-regulation of a locus containing putative nitrate assimilation genes, including the orphan response regulator nnaR (nitrate/nitrite assimilation regulator). These putative nitrate assimilation genes, narK3 (nitrate/nitrite transporter), nirBD (nitrite reductase), nnaR, and sirB (ferrochelatase) are arranged contiguously while nasN (assimilatory nitrate reductase identified in this work) is encoded in a different locus. Absence of a respiratory nitrate reductase in Mab and down-regulation of nitrogen metabolism genes in hypoxia suggest interplay between hypoxia adaptation and nitrate assimilation are distinct from what was previously documented in Mtb. The mechanisms used by Mab to fine-tune the transcriptional regulation of nitrogen metabolism in the context of stresses e.g. hypoxia, particularly the role of NnaR, remain poorly understood. To evaluate the role of NnaR in nitrate metabolism we constructed a Mab nnaR knockout strain (MabΔnnaR ) and complement (MabΔnnaR+C ) to investigate transcriptional regulation and phenotypes. qRT-PCR revealed NnaR is necessary for regulating nitrate and nitrite reductases along with a putative nitrate transporter. Loss of NnaR compromised the ability of Mab to assimilate nitrate or nitrite as sole nitrogen sources highlighting its necessity. This work provides the first insights into the role of Mab NnaR setting a foundation for future work investigating NnaR's contribution to pathogenesis.

Loss of LpqM proteins in Mycobacterium abscessus is associated with impaired intramacrophage survival.

Mycobacterium abscessus, an emerging pathogen responsible for severe pulmonary infections in cystic fibrosis patients, displays either a smooth (S) or a rough (R) morphotype. Infections with M. abscessus R are associated with increased pathogenicity in animal models and humans. While the S-to-R transition correlating with reduced glycopeptidolipid (GPL) production is well-documented, the recent screening of a transposon library revealed additional gene candidates located outside of the GPL locus involved in this transition. These genes include MAB_1470c, encoding the putative lipoprotein peptidase LpqM. However, experimental confirmation of the implication of this gene in the morphotype switch is lacking. Herein, we re-examined the role of MAB_1470c, and its homolog MAB_1466c, in colonial morphotype changes by generating unmarked deletion mutants in M. abscessus S. Our results indicate that the morphotype of these mutants stayed smooth in different media. Unexpectedly, the intracellular growth of ΔMAB_1470c and ΔMAB_1466c in THP-1 macrophages was significantly reduced as compared to the parental S strain, and these defects were rescued upon complementation with their corresponding genes. Strikingly, the intracellular survival defect was further exacerbated in a mutant lacking both MAB_1470c and MAB_1466c genes. This implies that, despite their primary sequence relatedness, the two proteins are not functionally redundant. Collectively, this suggests that these two LpqM-related lipoproteins are unlikely to be involved in the S-to-R transition but are key players for intramacrophage survival of M. abscessus. IMPORTANCE: Mycobacterium abscessus causes persistent infections in patients with underlying pulmonary diseases, resulting in progressive lung function deterioration. The rough (R) morphotype is well-established as associated with chronic and more aggressive infections in patients. In this study, we individually and simultaneously deleted the MAB_1470c and MAB_1466c genes in M. abscessus S, without observing changes in colony morphotypes. However, these mutants exhibited a severe impairment in their ability to survive within human macrophages, highlighting the critical role of these two lipoproteins in M. abscessus virulence.

Transcriptome profiles of macrophages upon infection by morphotypic smooth and rough variants of Mycobacterium abscessus.

Mycobacterium abscessus (Mab) infection can be deadly in patients with chronic lung diseases like cystic fibrosis (CF). In vitro and in vivo, Mab may adopt a smooth (S) or rough (R) morphotype, the latter linked to more severe disease conditions. In vitro studies revealed differences in pathogenicity and immune response to S and R morphotypes. We propose that in vivo both morphotypes exist and may transiently switch depending on the environment, having important pathogenic and immunologic consequences. This can be modeled by morphotypic S and R variants of Mab selected based on in vitro growth conditions. Here, we report the first analysis of early transcriptional events in mouse bone marrow derived macrophages (BMDMs) upon infection with media-selected interchangeable Mab-S and Mab-R morphotypes. The early transcriptional events after infection with both morphotypes showed considerable overlap of the pro-inflammatory genes that were differentially regulated compared to the uninfected macrophages. We also observed signature genes significantly differentially regulated in macrophages during infection of media-selected morphotypic Mab-S and Mab-R variants. In conclusion, media-selected Mab-S and Mab-R behave in a similar fashion to stable S and R types with respect to pathogenesis and immune response, serving as a useful model for environmentally influenced morphotype selection.

Bedaquiline susceptibility testing of Mycobacterium abscessus complex and Mycobacterium avium complex: A meta-analysis study.

OBJECTIVE: This study aims to estimate the overall in vitro activity of bedaquiline (BDQ) against clinical isolates of Mycobacterium abscessus complex (MABS) and M. avium complex (MAC), considering BDQ as a repurposed drug for non-tuberculous mycobacteria (NTM) infections. METHODS: We conducted a systematic review of publications in PubMed/ MEDLINE, Web of Science, and Embase up to 15 April 2023. Studies were included if they followed the Clinical and Laboratory Standards Institute (CLSI) criteria for drug susceptibility testing (DST). Using a random effects model, we assessed the overall in vitro BDQ resistance rate in clinical isolates of MABS and MAC. Sources of heterogeneity were analysed using Cochran's Q and the I2 statistic. All analyses were performed using CMA V3.0. RESULTS: A total of 24 publications (19 reports for MABS and 11 for MAC) were included. Using 1 µg/mL and 2 µg/mL as the breakpoint for BDQ resistance, the pooled rates of in vitro BDQ resistance in clinical isolates of MABS were found to be 1.8% (95% confidence interval [CI], 0.7-4.6%) and 1.7% (95% CI, 0.6-4.4%), respectively. In the case of MAC, the pooled rates were 1.7% (95% CI, 0.4-6.9%) and 1.6% (95% CI, 0.4-6.8%) for 1 µg/mL and 2 µg/mL, respectively. CONCLUSION: This study reports the prevalence of BDQ resistance in clinical isolates of MABS and MAC. The findings suggest that BDQ holds potential as a repurposed drug for treating MABS and MAC infections.

Noncanonical mutations in ribosome nascent peptide exit tunnel confer clarithromycin resistance in Mycobacterium abscessus complex.

OBJECTIVES: Mycobacterium abscessus is a non-tuberculous mycobacterial pathogen that causes pulmonary and skin infections globally. Clarithromycin plays a pivotal role in treating M. abscessus infections, with resistance often leading to treatment failure. While canonical mutations in the 23S rRNA residue 2270/2271 are recognized as the primary mechanism for acquired clarithromycin resistance, resistant isolates lacking these mutations have been widely reported. This study aims to identify new mechanisms of clarithromycin resistance in M. abscessus. METHODS: We selected spontaneous resistant mutants derived from two parental strains characterized by erm(41) T28 and C28 sequevars, respectively. Whole-genome sequencing was performed on mutants lacking the 23S rRNA 2270/2271 mutations. Site-directed mutagenesis was used to confirm the resistance phenotypes of newly identified mutations. Bioinformatic analysis of publicly available genomes was conducted to evaluate the presence of these mutations in clinical isolates. The spatial localization of these mutations in the ribosome was analyzed to investigate potential mechanisms of resistance. RESULTS: A total of 135 resistant mutants were selected from the parental strains. Sequencing of the 78 mutants lacking the 23S rRNA 2270/2271 mutations identified mutations within the peptidyl-transferase center and hairpin loops 35, 49, and 74 of the 23S rRNA. These noncanonical mutations were identified in 57 of 1875 genomes of clinical isolates. Thirteen representative mutations were introduced into the bacterial genome, and their contributions to macrolide resistance were confirmed. The newly identified mutations all localized at the entrance of the nascent peptide exit tunnel, potentially contributing to resistance by disrupting the macrolide binding pocket. CONCLUSION: Several noncanonical 23S rRNA mutations conferring clarithromycin resistance were identified. These mutations enhance our understanding of macrolide resistance in M. abscessus and could serve as important markers for diagnosing clarithromycin resistance.

Prevalence of resistance to macrolides and aminoglycosides in Mycobacterium avium, M. abscessus, and M. chelonae identified in the Laboratorio Nacional de Referencia of Colombia from 2018 to 2022 / Prevalencia de la resistencia a macrólidos y aminoglucósidos en los complejos Mycobacterium avium y M. abscessus y en Mycobacterium chelonae identificados en el Laboratorio Nacional de Referencia de Colombia entre el 2018 y el 2022.

Introduction: The Mycobacterium chelonae species and the M. avium and M. abscessus complexes are emerging pathogens that cause mycobacteriosis. Treatment depends on the species and subspecies identified. The drugs of choice are macrolides and aminoglycosides. However, due to the resistance identified to these drugs, determining the microbe's sensitivity profile will allow clinicians to improve the understanding of the prognosis and evolution of these pathologies. Objective: To describe the macrolide and aminoglycoside susceptibility profile of cultures identified by Colombia's Laboratorio Nacional de Referencia de Mycobacteria from 2018 to 2022, as Mycobacterium avium complex, M. abscessus complex, and M. chelonae. Materials and methods. This descriptive study exposes the susceptibility profile to macrolides and aminoglycosides of cultures identified as M. avium complex, M. abscessus complex, and M. chelonae using the GenoType® NTM-DR method. Materials and methods: This descriptive study exposes the susceptibility profile to macrolides and aminoglycosides of cultures identified as M. avium complex, M. abscessus complex, and M. chelonae using the GenoType® NTM-DR method. Results: We identified 159 (47.3 %) cultures as M. avium complex, of which 154 (96.9 %) were sensitive to macrolides, and 5 (3.1 %) were resistant; all were sensitive to aminoglycosides. From the 125 (37.2 %) cultures identified as M. abscessus complex, 68 (54.4 %) were sensitive to macrolides, 57 (45.6 %) were resistant to aminoglycosides, and just one (0.8 %) showed resistance to aminoglycosides. The 52 cultures (15.5 %) identified as M. chelonae were sensitive to macrolides and aminoglycosides. Conclusions: The three studied species of mycobacteria have the least resistance to Amikacin. Subspecies identification and their susceptibility profiles allow the establishment of appropriate treatment schemes, especially against M. abscessus.

Introducción. Mycobacterium chelonae y los complejos Mycobacterium avium y M. abscessus, son agentes patógenos emergentes causantes de micobacteriosis. El tratamiento de esta infección depende de la especie y la subespecie identificadas. Los fármacos de elección son los macrólidos y aminoglucósidos, contra los cuales se ha reportado resistencia; por esta razón, el determinar el perfil de sensibilidad le permite al médico tratante comprender mejor el pronóstico y la evolución de estas infecciones. Objetivo. Describir los perfiles de sensibilidad ante macrólidos y aminoglucósidos, de los cultivos identificados como complejo Mycobacterium avium, complejo M. abscessus o especie M. chelonae, en el Laboratorio Nacional de Referencia de Micobacterias durante los años 2018 a 2022. Materiales y métodos. Se llevó a cabo un estudio descriptivo del perfil de sensibilidad a macrólidos y aminoglucósidos, de los cultivos identificados como complejo M. avium, complejo M. abscessus o M. chelonae, mediante la metodología GenoType® NTM-DR. Resultados. Los cultivos del complejo M. avium fueron 159 (47,3 %), de los cuales, 154 (96,9 %) fueron sensibles y 5 (3,1 %) resistentes a los macrólidos; todos fueron sensibles a los aminoglucósidos. Del complejo M. abscessus se estudiaron 125 (37,2 %) cultivos, 68 (54,4 %) resultaron sensibles y 57 (45,6 %) resistentes a los macrólidos; solo un cultivo (0,8 %) fue resistente a los aminoglucósidos. De M. chelonae se analizaron 52 cultivos (15,5 %), todos sensibles a los macrólidos y aminoglucósidos. Conclusiones. En las tres especies de micobacterias estudiadas, la resistencia contra la amikacina fue la menos frecuente. La identificación de las subespecies y los perfiles de sensibilidad permiten instaurar esquemas de tratamiento adecuados, especialmente en las micobacteriosis causadas por M. abscessus.

Molecular Identification of Strains within the Mycobacterium abscessus Complex and Determination of Resistance to Macrolides and Aminoglycosides.

One of the most relevant and pathogenic groups among the rapidly growing mycobacteria (RGM) is Mycobacterium abscessus complex (MABC) that includes three subspecies: M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense. The aim of this study was the analysis of prevalence of MABC among other non-tuberculous mycobacteria isolated from patients in the Malopolska Region of Poland, between 2018 and 2021, as well as determination of their subspecies and molecular mechanisms of resistance to macrolides and aminoglycosides. The incidence of MABC was 5,4% (12/223). Eight strains were classified as M. abscessus subsp. abscessus, three as M. abscessus subsp. massiliense and one M. abscessus subsp. bolletii. Molecular analysis showed resistance to macrolides for eight strains of M. abscessus subsp. abscessus associated with erm(41)T28 gene mutations. One strain of M. abscessus subsp. abscessus showed resistance to macrolides (two mutations simultaneously: in erm(41)T28 and rrl genes) and aminoglycosides (point mutation in rrs gene). One strain of M. abscessus subs. bolletii was resistant to macrolides (erm(41)T28 mutation), whereas presented no mutations for aminoglycosides. M. abscessus subsp. massiliense reveal no mutations. High clarithromycin resistance of M. abscessus, determines the urgent need for susceptibility-based treatment. Molecular determination of resistance mechanisms to aminoglycosides and macrolides enables fast and accurate targeted treatment implementation.